DESCRIPTION:The long term objective of this project is to understand molecular mechanisms that regulate gene expression in nitrogen assimilation, including the roles of regulatory proteins, compartmentation of enzymes and substrates, and metabolite flux in the overall function of a metabolic pathway. Proline utilization in the yeast Saccharomyces cerevisiae serves as the model system for this study. In the wild, proline is the predominant nitrogen source for this organism. Its utilization requires the exhaustion of preferred, but less abundant, nitrogen sources and induction of the specific genes that encode the enzymes of this pathway. Induction is mediated by the Put3 protein, a transcriptional regulator that sits poised at the promoters of its target genes, remaining inactive unless proline is present. The focus of this study is to determine how Put3p is converted from an "off" to an "on" state and how it senses that proline is present and preferred nitrogen sources are absent from the growth medium. Two general models will be tested: a protein-protein or intermolecular interaction model and a conformational change or intramolecular interaction model. Put3p is modified by phosphorylation and ubiquitination; the role of each in the activity of the protein will be tested. Wild-type, constitutive and non-inducible put3 mutants will be examined for differences in modification. Pure Put3p will be analyzed by mass spectrometry to identify sites of modification. Limited proteolysis will be employed to determine if Put3p undergoes conformational changes in the presence of proline. The effect of proline on activation of Put3p is an in vitro transcription system will be tested. Put3p-interacting proteins will be sought using two-hybrid selections employing fragments of Put3p as the bait, and by the isolation of suppressors of Put3p constitutive mutants. Intramolecular interactions will be examined by a directed two-hybrid approach using fragments of Put3p as both bait and prey. The roles of the five characterized nitrogen repression regulators in the proline utilization system will be studied. This system provides a useful model to understand how changes in the cellular environment are signaled to the elements of a metabolism in humans. The work in Saccharomyces cerevisiae on proline metabolism contributed significantly to the recent isolation of human genes encoding homologous enzymes. Deficiencies in these enzymes cause hyperprolinemias; studies on the human genes will lead to a greater understanding of the molecular basis of these diseases.